Progress 04/09/17 to 03/16/22
Outputs
PROGRESS REPORT Objectives (from AD-416): The three objectives of this project are: (1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1]; (2) Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. [NP303, C2, PS2A]; and, (3) Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. Identify and characterize genes and/or phages affecting virulence and competitiveness of those bacteria to develop effective control methods. [NP303, C1, PS1]. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants (tested for absence of specific pathogens) for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Additional resources in the merged project will strengthen the research in the current Objective 1: Objective 1: Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1] Approach (from AD-416): The overall approach is to develop knowledge, tools, and reagents to aid U.S. floricultural producers and diagnosticians to establish and apply effective virus testing protocols to improve clean stock production for vegetatively-propagated annuals and perennials. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Based on the knowledge and tools developed while identifying and characterizing new viruses and comparisons to previously-characterized viruses, new virus-specific and broad spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, nucleic acid hybridization probes, PCR primers, isothermal amplification methods, and improved associated protocols will be developed. Validation of the recently devloped Universal Plant Virus Microarray (UPVM) will continue in order to transfer the UPVM technology to potential users. Next generation sequencing (NGS) of nucleic acid extracts from plants infected with unknown viruses is expected to yield information about the genomes of previously uncharacterized viruses without any background information on what viruses might be infecting the plant. Both NGS and UPVM have the potential to identify any virus present and identify all components of mixed infections, and is suited to application in situations where rapid results are important (in Quarantine operations and germplasm introduction). Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. We will make modifications to infectious clones of selected viruses by gene exchange and site-directed mutagenesis. We will examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST- pulldown assays. VIGS and/or protein over-expression will also be utilized. Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. The genomic DNA sequences of ornamental strains of Xylella fastidiosa (Xf) will be determined. The genetic diversity and phylogenetic relatedness among woody ornamental and non-ornamental strains will be evaluated. This sequence information will be used to develop specific PCR detection tools for woody ornamental strains of Xf. The identification and characterization of genes and regulatory elements, including phages, affecting virulence and/or competitiveness of Ralstonia solanacearum (including Race 3 Biovar 2) will be studied. This information will be used to further develop accurate detection tools and effective control methods. This is the final report for Project 8020-22000-042-000D, which ended in March 2022, and has been replaced by the new Project 8020-22000-052-000D, ⿿Detection, Biology, and Genomics of New and Emerging Viral and Bacterial Diseases of Ornamental Plants. Objective 1a: We examined diseased samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples included: a) bur oak (Quercus macrocarpa), having symptoms similar to those previously reported on common oak (Quercus robor) infected with an emaravirus in Germany, and currently under examination; b) two petunia samples showing a bright mosaic. Electron microscopy of the petunia samples revealed flexuous virions of c. 560-570 nm consistent with a potexvirus, and further characterization is in progress; c) Passiflora samples infected with a potyvirus; and d) Baptisia presumed to be infected with a potyvirus. We previously reported detection and identification in Maryland of an emaravirus infecting spicebush (Lindera benzoin), and tentatively named Lindera severe mosaic virus (LSMV). Seed of spicebush collected in the fall of 2021 and germinated in a greenhouse yielded a small number of seedlings with symptoms associated with LSMV infection. No spicebush plants were known to occur in the vicinity of the greenhouse, suggesting the possibility of seed transmission of LSMV. The presumed eriophyid mite vector (Phyllocoptes linderifolius) and symptoms were readily detected on leaves of spring 2022 growth of spicebush in the area where the seeds were collected, and mites were also observed on the immature fruit by a collaborating ARS mite expert. Experiments are underway to test the ability of the mite to transmit LSMV to healthy spicebush seedlings. Objective 1d: The complete genomes (RNAs 1-7) of two distinct rose rosette virus (RRV) isolates from the District of Columbia and Delaware were determined by high-throughput sequencing of total RNAs from symptomatic tissues. The complete genomes of two other co-infecting viruses (blackberry chlorotic ringspot virus and rose spring dwarf- associated virus) in the District of Columbia source were also determined. In initial collaborative RRV virus diversity studies with Texas A&M University colleagues, these RRV sequences were compared with 95 other RRV genomes deposited in the NCBI virus database (of which only three other isolates have their complete genomes determined). FY18-FY22: Substantial results were realized over the five years of the Project in each objective, which are summarized here. Objective 1: Determined the full genome sequences of three viruses co-infecting Crinum, Nerine latent carlavirus (and first report in the U.S.), Nerine yellow stripe potyvirus, and Crinum mosaic potyvirus (first report). Detection and full genome sequencing of two novel pelarspoviruses infecting star jasmine (Jasminum multiflorum) and angelwing jasmine (J. nitidum) plants from Hawaii, District of Columbia, Maryland, and California; and a third novel pelarspovirus in ornamental Clematis. Determined and first reported complete genome sequence of carnation latent virus, the type member of the genus Carlavirus. Detected and determined the near-complete sequences of isolates of apple mosaic virus and a novel carlavirus from plants of Magnolia tripetala. Determined the full sequence of an isolate of tulip virus X from Melissa officinalis. Developed virus-specific rabbit polyclonal and mouse monoclonal antibodies that can detect rose rosette virus, the rose rosette disease pathogen, in Western-blots and several ELISA formats. Determined the complete genomes (with 4 to 7 genome segments) of several new or emerging emaraviruses infecting landscape shrubs and trees, including beautyberry (Callicarpa americana), spicebush (Lindera benzoin), and rose (Rosa spp.). Determined the first full genome sequence of Helenium virus S from Veronica found in a mixed infection with two distinct isolates of Butterbur mosaic virus, one of which has a major deletion in an essential gene. Determined the near-complete genome sequence of two distinct isolates each of clover yellow mosaic virus and of white clover mosaic virus from co-infection in plants of while clover. Determined the near complete genomes of a novel allexivirus and an isolate of tobacco streak virus from a co-infected plant of Liriope muscari. Developed a highly specific and sensitive serological assay for the detection of Plantago asiatica mosaic virus in ornamental lily, which was applied for screening of multiple Asiatic, Oriental, and Tiger lilies. Demonstrated common occurrence of two non-vector eriophyid mites on roses and developed a high-resolution 3-D model of Phyllocoptes fructiphilus, the rosebud mite vector of rose rosette virus. Objective 2: Demonstrated that the interaction of Lolium latent virus major coat protein with a host ankyrin repeat protein NbANKr redirects it to chloroplasts and modulates virus infection. Determined viral sequence variations among 17 infectious clones of Turnip mosaic virus showing differential pathogenicity and infectivity in Nicotiana benthamiana, turnip, and Chinese cabbage. Developed chimeric infectious clones that affect symptoms and pathogenicity of Turnip mosaic virus; two determinants were shown to be required to induce stem necrosis in Nicotiana benthamiana, and to break resistance in Chinese cabbage. Determined the first sequences of Plantain virus X isolates and demonstrated that it is synonymous with the later-described Actinidia virus X and has a broader host range and geographic distribution than previously known. Demonstrated interactions between the dual coat proteins and the movement protein of Radish mosaic virus and their subcellular localization as free proteins and interacting aggregates. Demonstrated differential subcellular localization and RNA silencing efficiency of variants of the TGB1 protein of lily isolates of Plantago asiatica mosaic virus. Demonstrated that a single nucleotide change in the overlapping movement and coat protein genes of Youcai mosaic virus altering one amino acid residue in the coat protein caused milder symptoms. Objective 3: Developed and validated TaqMan-based real-time PCR assays for detection and differentiation of Ralstonia solanacearum select agent strains. Developed detection methods of select agent pathogen R. solanacearum race 3 biovar 2 with portable POCKIT and BLItz® systems. Identified a DNA region associated with cool virulence of R. solanacearum strain UW551 and developed the first PCR assay utilizing this region for specific detection of the select agent race 3 biovar 2 strains of R. solanacearum. Determined the molecular and biological characteristics of Rs551, a new filamentous bacteriophage isolated from a select agent race 3 biovar 2 strain of Ralstonia solanacearum. Discovered that prophage Rs551 and its repressor gene orf14 reduce virulence and increase completive fitness of its lysogenic select agent carrier strain UW551 of Ralstonia solanacearum. Discovered and determined the biological and molecular characteristics of four lytic Ralstonia phages isolated from Egypt, Indonesia, and the U.S. for their potential as biocontrol agents against Ralstonia solanacearum species complex strains. Identified and characterized a global LuxR-type transcriptional regulator, AclR, in Acidovorax citrulli, a seedborne bacterial pathogen responsible for bacterial fruit blotch. Discoved and determined the suggested significant role of the type III effector RipS1 in the cool virulence trait of the select agent Ralstonia solanacearum race 3 biovar 2 strains and as a potential target for the development of cool virulence-specific diagnostic tools to differentiate the highly regulated cool-virulent strains from non-cool-virulent strains of R. solanacearum. Developed specific detection and identification of American mulberry- infecting and Italian olive-associated strains of X. fastidiosa by FTP- LMAM (Fluorescence of TaqMan Probe upon Dequenching- Loop-Mediated Isothermal Amplification). ACCOMPLISHMENTS 01 Discovery of a jumbo Ralstonia-infecting phage isolated from the U.S. with promising biocontrol potential. The economically important bacterial wilt disease caused by R. solanacearum is difficult to control; using Ralstonia-infecting phages is a promising re-emerging control strategy. An ARS scientist in Beltsville, Maryland, isolated and characterized a jumbo phage that is the largest Ralstonia-infecting phage sequenced and reported to date. The phage has a wide host range, infecting each of the three newly established Ralstonia species in R. solanacearum species complex: R. solanacearum, R. pseudosolanacearum, and R. syzygii, and significantly reduced the virulence of a strain of R. solanacearum in tomato plants. This suggests that the jumbo phage has the potential to be developed into an effective control against diseases caused by R. solanacearum species complex strains. 02 Discovery of the type III effector RipS1 in the cool virulence of the select agent Ralstonia solanacearum race 3 biovar 2 strains. R. solanacearum race 3 biovar 2 strains cause brown rot of potato at cool temperatures and are listed as select agents in the U.S. and highly regulated. To gain a better understanding of cool-virulence mechanisms, ARS scientists in Beltsville, Maryland, generated libraries of transposon mutants in the cool virulent R. solanacearum strain UW551 and screened 10,000 mutants, using our previously published seedling assay, for significantly reduced virulence at cool, but not warm, temperatures. One mutant with the transposon altered the expression of a gene (RipS1) that may serve as a potential target for the development of cool virulence-specific diagnostic tools to differentiate the highly regulated cool-virulent strains from non-cool-virulent strains of R. solanacearum to safeguard U.S. agriculture.
Impacts
(N/A)
Publications
- Hammond, J. 2021. 2021 taxonomic update of phylum Negarnaviricota (Riboviria; Orthornavirae) including the large orders Bunyavirales and Mononegavirales. Archives of Virology. https://doi.org/10.1007/s00705-021- 05143-6.
- Hu, W., Seo, E., Cho, I., Kim, J., Song, Z., Kim, G., Eom, W., Jung, S., Hammond, J., Lim, H. 2022. Reassortment of infectious clones of radish mosaic virus shows that systemic necrosis in Nicotiana benthamiana is determined by RNA1. Phytopathology. https://doi.org/10.1094/PHYTO-04-21- 0172-R.
- Schachterle, J.K., Huang, Q. 2021. Implication of the type III effector RipS1 in the cool-virulence of Ralstonia solanacearum strain UW551. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2021.705717.
- Song, Z., Seo, E., Hu, W., Jeong, J., Moon, J., Kim, K., Eom, W., Cho, I., Hammond, J., Lim, H. 2022. Construction of full-length infectious cDNA clones of two Korean isolates of Turnip mosaic virus breaking resistance in Brassica napus. Archives of Virology. https://doi.org/10.1007/s00705- 022-05381-2.
- Song, Z., Chu, S., Hammond, J., Lim, H., Seo, E., Hu, W., Lim, Y., Park, T. , Park, J., Hong, J., Cho, I. 2022. Construction of full-length infectious clones of Turnip mosaic virus isolates infecting Perilla frutescens and genetic analysis of recently emerged strains of TuMV in Korea. Archives of Virology. https://doi.org/10.1007/s00705-021-05356-9.
- Inoue-Nagata, A., Jordan, R.L., Kreuze, J., Li, F., Lopez-Moya, J., Makinen, K., Ohshima, K., Wylie, S. 2022. ICTV Virus Taxonomy Profile: Potyviridae 2022. Journal of General Virology. 103:001738. https://doi.org/ 10.1099/jgv.0.001738.
- Cho, I., Chung, B., Yoon, J., Hammond, J., Lim, H. 2022. First report of Pepino mosaic virus infecting tomato in Korea. Plant Disease. https://doi. org/10.1094/PDIS-02-22-0380-PDN.
- Claros, N.A., Shires, M., Mollov, D.S., Hammond, J., Jordan, R.L., Ochoa- Corona, F., Olson, J., Ong, K., Salamanca, R. 2022. Rose Rosette Disease: A Diagnostic Guide. Plant Health Progress. https://doi.org/10.1094/PHP-05- 22-0047-DG.
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): The three objectives of this project are: (1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1]; (2) Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. [NP303, C2, PS2A]; and, (3) Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. Identify and characterize genes and/or phages affecting virulence and competitiveness of those bacteria to develop effective control methods. [NP303, C1, PS1]. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants (tested for absence of specific pathogens) for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Additional resources in the merged project will strengthen the research in the current Objective 1: Objective 1: Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1] Approach (from AD-416): The overall approach is to develop knowledge, tools, and reagents to aid U.S. floricultural producers and diagnosticians to establish and apply effective virus testing protocols to improve clean stock production for vegetatively-propagated annuals and perennials. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Based on the knowledge and tools developed while identifying and characterizing new viruses and comparisons to previously-characterized viruses, new virus-specific and broad spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, nucleic acid hybridization probes, PCR primers, isothermal amplification methods, and improved associated protocols will be developed. Validation of the recently devloped Universal Plant Virus Microarray (UPVM) will continue in order to transfer the UPVM technology to potential users. Next generation sequencing (NGS) of nucleic acid extracts from plants infected with unknown viruses is expected to yield information about the genomes of previously uncharacterized viruses without any background information on what viruses might be infecting the plant. Both NGS and UPVM have the potential to identify any virus present and identify all components of mixed infections, and is suited to application in situations where rapid results are important (in Quarantine operations and germplasm introduction). Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. We will make modifications to infectious clones of selected viruses by gene exchange and site-directed mutagenesis. We will examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST- pulldown assays. VIGS and/or protein over-expression will also be utilized. Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. The genomic DNA sequences of ornamental strains of Xylella fastidiosa (Xf) will be determined. The genetic diversity and phylogenetic relatedness among woody ornamental and non-ornamental strains will be evaluated. This sequence information will be used to develop specific PCR detection tools for woody ornamental strains of Xf. The identification and characterization of genes and regulatory elements, including phages, affecting virulence and/or competitiveness of Ralstonia solanacearum (including Race 3 Biovar 2) will be studied. This information will be used to further develop accurate detection tools and effective control methods. Under Objective 1a: We examined samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples included: a) plants of Lindera benzoin from a Maryland nursery, showing a strong foliar mosaic and distortion, which was determined by reverse transcriptase-polymerase chain reaction assay to be infected with an emaravirus recently sequenced by a collaborating ARS scientist at Beltsville, Maryland, in samples with similar symptoms from Indiana and Maryland, and also detected in Kentucky, Missouri, Ohio, and Virginia; the virus has been tentatively named Lindera severe mosaic virus, and in collaboration with an ARS mite expert, the putative eriophyid mite vector found on infected plants was identified as Phyllocoptes linderafolius. b) plants of Callicarpa americana showing foliar mosaic were shown to be infected with Constricta yellow dwarf virus (previously known as Potato yellow dwarf virus-CYDV). This is new host for this virus, which mainly infects solanaceous plants and a few herbaceous hosts. Under Objective 1d: In collaboration with other ARS colleagues at Beltsville, Maryland, several samples were subjected to high-throughput sequencing, including: a) a composite sample of Liriope muscari showing chlorotic or whitish foliar streaking, in which long flexuous virus-like particles were previously revealed by electron microscopy, yielded near complete sequences of a novel allexivirus, and of tobacco streak virus; b) a sample originating from clover, and subsequently propagated in Nicotiana benthamiana. This was previously determined to be infected by a potexvirus, but high throughput sequencing revealed the presence of a complex mixed infection of two distinct isolates of clover yellow mosaic virus and also two distinct isolates of white clover mosaic virus; c) samples of Magnolia tripetala from Massachusetts were similar to nearly complete sequences of either apple mosaic virus (previously detected in a sample from Pennsylvania) or a carlavirus closely related to isolates previously detected in a sample from Virginia, and to the full sequence previously identified by a colleague from the University of Mississippi in a sample from North Carolina; d) a plant of Rehmannia angulata showing an obvious mosaic was found to be infected with an isolate of cucumber mosaic virus; e) samples from other Callicarpa americana plants showing foliar mosaic were not found to be infected with either of two novel emaraviruses already under study, but were shown to be infected with a novel badna virus; and, f) in partnership with Korean collaborators, three new virus:crop combinations were detected for the first time in Korea - Passiflora latent virus was identified infecting persimmon (Diospyros kaki), Tomato mosaic virus in chili pepper (Capsicum annuum), and Cucurbit chlorotic yellows virus in muskmelon and Oriental melon (Cucumis melo). Under Objective 2a: In partnership with Korean collaborators, full length infectious clones of three isolates of radish mosaic virus differing in host responses were developed and pseudo-recombinants generated by inoculating groups of plants with RNA1 of each isolate separately with RNA2 of each of the three isolates. By this means it was shown that induction of systemic necrosis in Nicotiana benthamiana is determined by RNA1 of isolates A or G, whereas severe mosaic, necrotic ringspots, and veinal necrosis in radish was determined by RNA1 of isolate B. Thus isolates A or G and showed reciprocal results to isolate B for symptom severity and systemic necrosis between N. benthamiana and radish. Japanese scientists had previously implicated an amphipathic helix in the helicase protein encoded by RNA1 as the inducer of necrosis in N. benthamiana, but the amino acid sequence of this helix is identical in the helicase proteins of the three Korean isolates, suggesting that a differential interaction with host proteins of N. benthamiana and radish determined by another region of the helicase is responsible for the reciprocal symptom difference in these two hosts. Record of Any Impact of Maximized Teleworking Requirement: FY2020 and FY2021 Maximized Telework minimized the amount of lab work that could be completed and limited interactions with colleagues. For example, the limited interactions with colleagues reduced our ability to perform confocal microscopy, which was also compounded by illness and passing of the Director of the Microscopy Unit. ACCOMPLISHMENTS 01 Development of a high-throughput virulence screening method for Ralstonia solanacearum species complex strains. R. solanacearum species complex strains are the causative agents for wilting diseases of many plants, including the economically important brown rot of potato. ARS scientists at Beltsville, Maryland, developed a high-throughput virulence screen implemented in 96-well microtiter plates using Nicotiana glutinosa grown in soft water agar which saves space, time, and resources. This assay can be used to rapidly screen many strains, isolates, or mutants for disease symptoms under both cool (20C) and warm (28C) temperature conditions to narrow down isolates for more detailed testing. This assay provides a valuable tool for future work in understanding genetics of virulence of R. solanacearum species complex strains, especially the cool virulence of the select agent race 3 biovar 2 group of R. solanacearum, leading toward development of effective control strategies. 02 A sensitive and specific tool to detect and identify strains of Xylella fastidiosa infecting American mulberry and Italian olive. The bacterium X. fastidiosa causes serious plant diseases in many important agricultural crops and landscape trees. In Italy, it was identified for the first time in 2013 in olive trees affected by a devastating disease denoted ⿿olive quick decline syndrome⿿. Recently, it has also been found in other European countries including Germany, France, Spain, and Portugal. An ARS scientist in Beltsville, Maryland, collaborated with scientists in Italy to develop TaqMan-based qPCR, Loop-mediated isothermal amplification (LAMP), and Fluorescence of TaqMan Probe upon Dequenching - Loop-mediated Isothermal Amplification (FTP-LAMP) assays based on a unique gene identified by the ARS scientist. The new FTP- LAMP assay is highly sensitive, detecting down to 100 fg genomic DNA of X. fastidiosa, and highly specific (98.7% vs 89% for other LAMP-based assays). Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP assay represents a useful tool not only for the specific detection of the olive-associated strain in Italy, but also in differentiating the Italian De Donno strain from other strains of X. fastidiosa already reported in Italy, Germany, France, Spain, and Portugal. 03 First complete genome sequence of the genus Carlavirus type species, Carnation latent virus. The International Committee on Taxonomy of Viruses (ICTV) is charged with the task of developing, refining, and maintaining a universal virus taxonomy. Carlavirus is a genus of plant viruses that includes 53 species recognized by ICTV. Molecular sequences of many of these species are well-represented in the NCBI GenBank database. However, surprisingly, the type species for this genus, Carnation latent virus (CLV), had no full-length genomic sequences in the database. ARS scientists in Beltsville, Maryland, determined the full-length genome sequence of CLV using high-throughput sequencing. The complete sequence was determined to be 8,513 nucleotides and was deposited in GenBank. The sequence data and phylogenetic analysis confirm that carnation latent virus is distinct from all other recognized carlaviruses. This full genome sequence of Carnation latent virus will now serve as the Genome Reference Sequence to which all other carlaviruses are compared. 04 Discovery of a Streptomyces scabies-infecting bacteriophage from Egypt with promising biocontrol traits. Potato common scab caused by Streptomyces scabies is one of the most economically important diseases infecting potato. Biological control using bacteriophages is a promising strategy for controlling this disease. An ARS scientist in Beltsville, Maryland, in collaboration with scientists in Egypt, discovered and characterized a novel bacteriophage (designated SscP1EGY) isolated from a potato field in Egypt that showed high lytic efficacy against seven tested S. scabies strains, but showed no lytic activity against three beneficial Streptomyces species, other beneficial bacterial species, and non-target plant pathogenic bacteria. In greenhouse experiments, treatment of S. scabies-inoculated potato tubers with phage SscP1EGY resulted in superficial rather than pitted scab lesions, and reductions in the number of lesions and overall severity of scab, compared to inoculated tubers without phage treatment. These results suggest that SscP1EGY has potential as a biological control agent for this economically important pathogen. 05 Identification and characterization of a global regulator, AclR, in Acidovorax citrulli. LuxR-type transcriptional regulators are essential for many physiological processes in bacteria, including pathogenesis. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes great losses in melon and watermelon worldwide. However, the LuxR-type transcriptional factors in A. citrulli have not been well studied, except the previously reported LuxR-type regulatory protein, AcrR, involved in regulating virulence and motility. Scientists in China, in collaboration with an ARS scientist in Beltsville, Maryland, characterized a second LuxR-type regulator, AclR, in the group II strain Aac-5 of A. citrulli by mutagenesis and transcriptomic analysis. They found that AclR plays a global role in transcriptional regulation in A. citrulli influencing motility, biofilm formation, and virulence, and provides a new perspective regarding the regulatory network of biological functions in A. citrulli. Increased understanding of the molecular mechanisms behind the regulation of A. citrulli by AclR will facilitate the development of effective control strategies to combat this important bacterial pathogen. 06 Genome sequences of two distinct carlaviruses from a mixed infection in veronica. Mixed infections may result in either more severe or attenuated symptoms compared to single infections. A Veronica hybrid showing foliar mosaic and distortion was previously found by ARS researchers in Beltsville, Maryland, to be infected by a mixture of two distinct carlaviruses. Near-complete sequences of both butterbur mosaic virus (ButMV) and helenium virus S (HelVS) revealed that two distinct isolates of ButMV were present, one of which was defective, having a deletion of almost the complete sequence of a gene normally considered essential; this is the first example of a defective isolate of a carlavirus being maintained in the viral population through complementation by the fully functional ButMV isolate. The sequence of HelVS is the first near-complete genome of any HelVS isolate. This work expands the known occurrence of viruses in this perennial flowering ornamental; both viruses are transmitted by aphids and have restricted natural host ranges; ButMV has previously been reported only from Japanese butterbur (Petasites japonica) in Japan and South Korea, while HelVS has previously been reported only from Helenium amarum and Impatiens holstii in Europe and the USA. Testing of stock plants of veronica prior to mass propagation is suggested to minimize the distribution of these viruses in nursery-grown plants.
Impacts
(N/A)
Publications
- Hammond, J., Adams, I., Fowkes, A.R., Mcgriegs, S., Botermans, M., Von Oorspronk, J., Westenberg, M., Verbeek, M., Dullemans, A., Stijger, C., Blouin, A., Massart, S., De Jonghe, K., Heyneman, M., Walsh, J.A., Fox, A. 2020. Sequence analysis of 43-year old samples of Plantago lanceolata show that Plantain virus X is synonymous with Actininidia virus X and is widely distributed. Plant Pathology. 70(2):249-258. https://doi.org/10.1111/ppa. 13310.
- Hammond, J. 2020. 2020 taxonomic update for phylum Negarnaviricota (Riboviria; Orthornavirae) including the large orders Bunyavirales and Mononegavirales. Archives of Virology. https://doi.org/10.1007/s00705-020- 04731-2.
- Cho, I., Yang, C., Yoon, J., Kwon, T., Hammond, J., Lim, H. 2021. First report of Passiflora latent virus infecting persimmon (Diospyros Kaki) in Korea. Plant Disease. 105(4):1236. https://doi.org/10.1094/PDIS-07-20-1502- PDN.
- Cho, I., Yoon, J., Yang, C., Chae, S., Chung, B., Hammond, J., Lim, H. 2021. First report of Tomato mosaic virus in chili pepper in Korea. Journal of Plant Pathology. https://doi.org/10.1007/s42161-021-00854-w.
- Abrahamian, P., Hammond, J., Hammond, R. 2021. Development and optimization of a Pepino mosaic virus-based vector for rapid expression of heterologous proteins in plants. Applied Microbiology and Biotechnology. 105:627-645. https://doi.org/10.1007/s00253-020-11066-0.
- Elbeaino, T., Incerti, O., Dakroub, H., Valentini, F., Huang, Q. 2020. Exploiting the real-time PCR and LAMP for development of a conjunctive new assay (FTP-LAMP) for the specific detection and differentiation of Xylella fastidiosa De Donno and mulberry strains from other subspecies/strains..... . Journal of Microbiological Methods. https://doi.org/10.1016/j.mimet. 2020.105992.
- Stommel, J.R., Dumm, J.M., Hammond, J. 2021. Effect of ozone on inactivation of purified pepper mild mottle virus and contaminated pepper seed. Phytofrontiers. 1: 85-93. https://doi.org/10.1094/PHYTOFR-09-20-0020- R.
- Jordan, R.L., Korolev, E., Grinstead, S.C., Mollov, D.S. 2021. First complete genome sequence of carnation latent virus, the type member of the genue Carlavirus. Archives of Virology. 166:1501-1505. https://doi.org/10. 1007/s00705-021-04999-y.
- Schachterle, J.K., Huang, Q. 2021. A high-throughput virulence screening method for the Ralstonia solanacearum species complex. Journal of Microbiological Methods. https://doi.org/10.1016/j.mimet.2021.106270.
- Hammond, J., Reinsel, M.D., Grinstead, S.C., Lockhart, B., Jordan, R.L., Mollov, D.S. 2020. A mixed infection of helenium virus S with two distinct isolates of butterbur mosaic virus, one of which has a major deletion in an essential gene. Frontiers in Microbiology. https://doi.org/10.3389/ fmicb.2020.612936.
- Scheets, K., Jordan, R.L. 2021. Create two new species in genus Pelarspovirus (Tolivirales: Tombusviridae). Electronic Publication. https:/ /doi.org/.
- Cho, I., Kim, T., Yoon, J., Chung, B., Hammond, J., Lim, H. 2021. First report of Cucurbit chlorotic yellows virus infecting Cucumis melo (muskmelon and oriental melon) in Korea. Plant Disease. https://doi.org/10. 1094/PDIS-11-20-2375-PDN.
- Abdelrhim, A.S., Omar, M.O., Hammad, A.M., Ahmad, A.A., Huang, Q. 2021. A new Streptomyces scabies-infecting bacteriophage from Egypt with promising biocontrol traits. Archives Of Microbiology. https://doi.org/10.1007/ s00203-021-02415-2.
- Guan, W., Wang, T., Zhao, M., Huang, Q., Tian, E., Liu, Y., Liu, B., Yang, Y., Zhao, T. 2021. Transcriptomic and functional analyses reveal roles of AclR, a luxR-type global regular in regulating motility and virulence of Acidovorax citrulli. Molecular Plant-Microbe Interactions. https://doi.org/ 10.1094/MPMI-01-21-0020-R.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): The three objectives of this project are: (1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1]; (2) Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. [NP303, C2, PS2A]; and, (3) Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. Identify and characterize genes and/or phages affecting virulence and competitiveness of those bacteria to develop effective control methods. [NP303, C1, PS1]. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants (tested for absence of specific pathogens) for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Additional resources in the merged project will strengthen the research in the current Objective 1: Objective 1: Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1] Approach (from AD-416): The overall approach is to develop knowledge, tools, and reagents to aid U.S. floricultural producers and diagnosticians to establish and apply effective virus testing protocols to improve clean stock production for vegetatively-propagated annuals and perennials. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Based on the knowledge and tools developed while identifying and characterizing new viruses and comparisons to previously-characterized viruses, new virus-specific and broad spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, nucleic acid hybridization probes, PCR primers, isothermal amplification methods, and improved associated protocols will be developed. Validation of the recently devloped Universal Plant Virus Microarray (UPVM) will continue in order to transfer the UPVM technology to potential users. Next generation sequencing (NGS) of nucleic acid extracts from plants infected with unknown viruses is expected to yield information about the genomes of previously uncharacterized viruses without any background information on what viruses might be infecting the plant. Both NGS and UPVM have the potential to identify any virus present and identify all components of mixed infections, and is suited to application in situations where rapid results are important (in Quarantine operations and germplasm introduction). Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. We will make modifications to infectious clones of selected viruses by gene exchange and site-directed mutagenesis. We will examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST- pulldown assays. VIGS and/or protein over-expression will also be utilized. Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. The genomic DNA sequences of ornamental strains of Xylella fastidiosa (Xf) will be determined. The genetic diversity and phylogenetic relatedness among woody ornamental and non-ornamental strains will be evaluated. This sequence information will be used to develop specific PCR detection tools for woody ornamental strains of Xf. The identification and characterization of genes and regulatory elements, including phages, affecting virulence and/or competitiveness of Ralstonia solanacearum (including Race 3 Biovar 2) will be studied. This information will be used to further develop accurate detection tools and effective control methods. Under Objective 1a: We examined diseased samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples include: i) a plant of Phlox caroliniana previously found to be infected with a potyvirus was further shown to also be infected with cucumber mosaic virus (CMV), confirmed by RT-PCR and sequencing; ii) plants of honeysuckle (Lonicera sempervirens and two named hybrids) with poor rooting success were examined. One hybrid was found to be infected with a probable carlavirus (slightly flexuous particles of 620-640 nm), for which further characterization is in progress; iii) foliage of Southern hackberry (Celtis laevigata) exhibiting decline after development of generalized mottle and chlorosis. Electron microscopy of sections revealed presence of many fine filaments, and multiple membranous abnormalities in the cytoplasm of affected leaves; iv) multiple samples of lilyturf (Liriope muscari) with chlorotic to white streaking on the foliage were obtained from different sources. Electron microscopy revealed flexuous particles of c.800 nm, and further characterization is on-going; v) plants of garlic mustard (Alliaria petiolata) with obvious mosaic were found to be infected with a presumed potyvirus, for which further characterization is in progress; vi) plants of closed bottle gentian (Gentiana andrewsii) showing necrotic spotting and some veinal necrosis were infected with a putative potyvirus, which is undergoing further characterization. Under Objective 1d: We determined the full-length genome of Carnation latent virus (CLV), which is the type species for the Carlavirus genus. The complete CLV-KL sequence was determined to be 8,513 nt using high- throughput sequencing and was validated by Sanger sequencing of CLV- specific RT-PCR generated amplicons and 5' and 3' RACE protocols. In pairwise analysis, the genome shares 40-46% identity with other recognized carlaviruses and the six in silico translated proteins have 15- 62% amino acid identities with their respective carlavirus orthologs. In phylogenetic analysis, CLV clusters with Butterbur mosaic virus, Coleus vein necrosis virus, and Garlic common latent virus. The CLV-KL coat protein shares 98% identity with the CLV-UK NCBI Reference Sequence. In collaboration with Korean colleagues, Pyrus pyrifolia cryptic virus was identified infecting pear trees in Korea; no specific symptoms could be attributed to this virus, as the infected trees had mixed infections with up to four viruses commonly known to infect pear. Work continued on historic and contemporary isolates of plantain virus X (PlVX) with collaborators in the United Kingdom (UK) and the Netherlands, and expanded to include additional colleagues in the Netherlands and Belgium. This expanded collaboration has resulted in determination of essentially full genome sequences from two historic PlVX isolates from Plantago lanceolata in the UK, one from P. lanceolata in the Netherlands, one from Capsicum annuum originating from Ethiopia, and one from Browallia americana in Belgium, plus multiple additional partial sequences from the UK, the Netherlands, and Belgium. In addition to the previous determination that plantain virus X is synonymous with actinidia virus X (AVX; reported in New Zealand from Actinidia chinensis imported from China, and in Canada from Ribes nigrum imported from an undisclosed source), it is now clear that PlVX/AVX has a natural host range including plants from at least four taxonomically diverse plant families (the Plantaginaceae, Solanaceae, Actinidiaceae, and Grossulariaceae), and has an apparent geographic range including the UK, the Netherlands, Belgium, Ethiopia, and China, with documented importations into New Zealand and Canada. Under Objective 2a: In partnership with Korean collaborators, full- length infection clones of two additional Korean isolates of turnip mosaic virus (TuMV) were generated. Isolate TuMV-KBC belongs to the World- B phylogenetic group of isolates, and infects Chinese cabbage but not radish; in contrast, isolate TuMV-KRS belongs to the Basal-BR phylogenetic group, and infects radish but not Chinese cabbage. Chimeric infectious clones were prepared by exchanging an internal (~48%) portion of the genomes of isolates KBC and KRS in order to determine which viral genes control host range; additional chimeric constructs exchanging smaller fragments of the viral genome are currently in progress to more finely map the viral determinants of host range specificity. In collaboration with Korean colleagues, the full sequence of a Korean isolate of radish mosaic virus (RaMV), only recently reported in Korea, was determined. Subsequently, infectious clones of the two genomic segments of this RaMV isolate were developed, and modified by insertion of a unique restriction enzyme site into the 3⿿-terminal untranslated region of the infectious clone of RNA 2, to allow use as a Virus-Induced Gene Silencing (VIGS) vector for cruciferous crops. A fragment of the phytoene desaturase gene was inserted to demonstrate the efficacy of the modified RaMV as a VIGS vector, resulting in the expected development of chlorotic bleaching of leaf tissue. This RaMV-derived VIGS vector will be further tested to aid in determining the function of host genes of radish and other field crops and ornamental species of cruciferous crops. In collaboration with other ARS colleagues at the Beltsville location, the complete sequence of a Maryland isolate of pepino mosaic virus (PepMV) was determined, and shown to belong to the ⿿European⿿ clade of PepMV isolates. Infectious clones were subsequently developed, and variants prepared to express the gene for the green fluorescent protein (GFP) as a marker to track systemic infection of inoculated plants. Under Objective 3.1: In collaboration with scientists in Italy, a gene sequence that we previously identified that is uniquely present in Italian olive and American mulberry strains of Xylella fastidiosa was used to design primers and probe for the development of real-time detection methods for these Italian olive and American mulberry strains. These methods included TaqMan qPCR and LAMP assays, and a new technology, ⿿Fluorescence of TaqMan Probe upon Dequenching -Loop-Mediated Isothermal Amplification (FTP-LAMP) assay. Under Objective 3.2a: We isolated a 'jumbo' R. solanacearum-infecting phage from soil in the United States and characterized its genome. We also determined the morphology, growth characteristics, thermal stability, host range, and effect on the virulence of its host bacterial strain. Under Objective 3.2b: We performed genetic screens to identify temperature-responsive regulatory elements in cool-virulent R. solanacearum by random mutagenesis of the cool-virulent R. solanacearum strain UW551 with a transposon carrying a promoter-less bacterial luciferase (luxCDABE) operon as a reporter. We also screened the transposon mutants at cool (20oC) and tropical (28oC) temperatures and identified specific mutants in which expression of the reporter is temperature-dependent, indicating that the transposon inserted downstream of a temperature-dependent promoter. We also developed high-throughput R. solanacearum virulence screening methods using tobacco seedlings and used this method to screen random transposon mutants of the cool virulent strain UW551 of R. solanacearum to identify mutants that lost virulence at cool but not at high temperatures, in order to study mechanisms that differentiate cool-virulent strains from tropical strains of in causing disease at cool temperature. Accomplishments 01 Identification and characterization of a global regulator, AcrR, in Acidovorax citrulli. Identification and characterization of a global regulator, AcrR, in Acidovorax citrulli. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes significant losses in melon and watermelon worldwide. Bacterial motility and virulence in this and other species are controlled by LuxR-type regulators. ARS scientists in Beltsville, Maryland, collaborated with scientists in China to identify a LuxR-type regulator, AcrR, in a strain of A. citrulli, Aac-5. They found that the acrR mutant had reduced virulence and lost twitching and swimming motilities and flagellar formation, but increased biofilm formation and growth ability. This new knowledge indicates that AcrR acts as a global regulator affecting multiple important biological functions of A. citrulli, and should lead to the development of effective control strategies to combat this economically important bacterial pathogen.
Impacts
(N/A)
Publications
- Choi, G., Cho, I., Ju, H., Hu, W., Kim, B., Oh, J., Seo, E., Park, J., Domier, L.L., Lim, H., Hammond, J., Song, K. 2019. Full-length infectious clones of two new isolates of tomato mosaic virus induce distinct symptoms associated with two differential amino acid residues in 128 kDa protein. Plant Pathology Journal. 35(5):538-542.
- Guan, W., Wang, T., Huang, Q., Tian, E., Liu, B., Yang, Y., Zhao, T. 2020. A LuxR-type regulator, AcrR, regulates flagellar assembly and contributes to virulence, motility, biofilm formation and growth ability of acidovorax citrulli. Molecular Plant Pathology. 00:1-13.
- Hu, W., Kim, B., Kwak, Y., Seo, E., Kim, J., Han, J., Kim, I., Lim, Y., Cho, I., Domier, L.L., Hammond, J., Lim, H. 2019. Five newly collected turnip mosaic virus (TuMV) isolates from Jeju Island, Korea are closely related to previously reported Korean TuMV isolates but show distinctive symptom development. Plant Pathology Journal. 35(4):381-387.
- Oh, J., Choi, G., Kim, J., Oh, M., Kim, K., Park, J., Domier, L.L., Hammond, J., Lim, H. 2019. Differences in isolates of tomato yellow leaf curl virus in tomato fields located in Daejeon and Chungcheongnam-do between 2017 and 2018. Korean Journal of Agricultural Science. 46(3):507- 517.
- Cho, I., Yang, C., Kwon, S., Yoon, J., Kim, D., Choi, G., Hammond, J., Moon, J., Lim, H. 2019. First report of Grapevine Syrah virus 1 infecting grapevines in Korea. Plant Disease. 103(11):29-70.
- Abrahamian, P., Hammond, J., Hammond, R. 2020. Complete genome sequence of an American isolate of Pepino mosaic virus. Microbiology Resource Announcements. 9/e01124-19.
- Jordan, R.L., Hammond, J. 2020. Bean common mosaic virus and bean common mosaic necrosis virus (Potyviridae). Encyclopedia of Virology.
- Kreuze, J.F., Vaira, A., Menzel, W., Candresse, T., Zariev, S.K., Hammond, J., Ryu, K., Report Consortium, I. 2020. ICTV Virus Taxonomy Profile: Alphaflexiviridae. Journal of General Virology.
- Cho, I., Yang, C., Kwon, S., Yoon, J., Kwon, T., Hammond, J., Lim, H. 2020. First report of Pyrus pyrifolia cryptic virus infecting pear in Korea. Plant Disease.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): The three objectives of this project are: (1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1]; (2) Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. [NP303, C2, PS2A]; and, (3) Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. Identify and characterize genes and/or phages affecting virulence and competitiveness of those bacteria to develop effective control methods. [NP303, C1, PS1]. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants (tested for absence of specific pathogens) for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Approach (from AD-416): The overall approach is to develop knowledge, tools, and reagents to aid U.S. floricultural producers and diagnosticians to establish and apply effective virus testing protocols to improve clean stock production for vegetatively-propagated annuals and perennials. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Based on the knowledge and tools developed while identifying and characterizing new viruses and comparisons to previously-characterized viruses, new virus-specific and broad spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, nucleic acid hybridization probes, PCR primers, isothermal amplification methods, and improved associated protocols will be developed. Validation of the recently devloped Universal Plant Virus Microarray (UPVM) will continue in order to transfer the UPVM technology to potential users. Next generation sequencing (NGS) of nucleic acid extracts from plants infected with unknown viruses is expected to yield information about the genomes of previously uncharacterized viruses without any background information on what viruses might be infecting the plant. Both NGS and UPVM have the potential to identify any virus present and identify all components of mixed infections, and is suited to application in situations where rapid results are important (in Quarantine operations and germplasm introduction). Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. We will make modifications to infectious clones of selected viruses by gene exchange and site-directed mutagenesis. We will examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST- pulldown assays. VIGS and/or protein over-expression will also be utilized. Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. The genomic DNA sequences of ornamental strains of Xylella fastidiosa (Xf) will be determined. The genetic diversity and phylogenetic relatedness among woody ornamental and non-ornamental strains will be evaluated. This sequence information will be used to develop specific PCR detection tools for woody ornamental strains of Xf. The identification and characterization of genes and regulatory elements, including phages, affecting virulence and/or competitiveness of Ralstonia solanacearum (including Race 3 Biovar 2) will be studied. This information will be used to further develop accurate detection tools and effective control methods. We examined samples of various ornamentals from plant disease clinics, nurseries, or individuals, including: i) a plant of Veronica found to be infected by Helenium virus S as with prior plants of three cultivars; ii) plants of Phlox maculata x Phlox glabberima, and Phlox carolina showing necrotic streaking and etching of leaves - electron microscopy revealed pinwheel cytoplasmic inclusions typical of potyviruses; iii) plants of Coreopsis showing brown leaf streaking - a putative potyvirus was detected by PCR using generic potyvirus primers. In collaboration with scientists from the Systematic Entomology Laboratory and the Electron and Confocal Microscopy Unit at Beltsville, Maryland, we continue to examine interactions between eriophyid mites and different rose species or genotypes received from collaborators under a USDA-NIFA-SCRI project to examine the viral, vector, and host factors affecting spread of Rose rosette virus and the associated disease in roses. Collaboration was initiated with scientists from USDA-APHIS and PathSensors, Inc. (Baltimore, Maryland) to develop improved protocols for detection of potyviruses using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) biosensor system. Several years ago, the USDA-ARS Beltsville Lab developed a hybridoma cell line that secretes a broad-spectrum reacting PTY-1 monoclonal antibody (McAb). This hybridoma and McAb, which detects almost all known aphid-transmitted potyviruses, was patented and licensed to Agdia, Inc., and forms the basis of Agdia⿿s ⿿POTY Group Test reagents, ELISA kits, and ImmunoStrip Test. CANARY is a fast, sensitive, and easy-to-use biosensor-based technology developed at MIT that has been successfully utilized for the detection of bacteria, viruses, and toxins. Several PTY-1 hybridoma- derived POTY CANARY biosensor clones were created at MIT-LL, initially tested in the APHIS-CPHST Beltsville Lab, and later evaluated collaboratively in the USDA-ARS Beltsville and PathSensors labs, for assay feasibility, bead capture efficacy, specificity and sensitivity of the reaction to detect target potyviruses. We have optimized the POTY biosensor assay and shown that it can detect diverse purified potyviruses down to less than 5 ng/ml and can detect multiple potyviruses in naturally infected sources, such as various isolates of potato virus Y in potato leaves and tubers. We continue to utilize broad-spectrum PCR primers for analysis of a wide range of suspected virus-infected plants, allowing detection of previously reported viruses in new hosts, or obtaining products for sequence analysis of newly-discovered viruses allowing their comparison to previously characterized viruses. Using generic primers capable of amplifying products from multiple potyviruses, or both potexviruses and carlaviruses, products were obtained from samples including: Coreopsis (an unidentified potyvirus), Veronica (Helenium virus S, a carlavirus), and bamboo (Bamboo mosaic virus, a potexvirus). In our ongoing research on the development of broad-spectrum reacting antibodies to important plant virus species, additional potexvirus and carlavirus sequences have been cloned for bacterial expression of the coat protein sequences to aid in the analysis of cross-reactive rabbit polyclonal (PcAb) and mouse monoclonal antibodies (McAbs) recently generated to synthetic peptide immunogens containing highly conserved regions of carlavirus or potexvirus coat proteins. Carlavirus peptide- specific PcAbs were screened against the immunogen peptides, virions and cloned CPs from more than 20 diverse carlaviruses and were shown to react with multiple carlaviruses. Eighty newly developed hybridoma cell lines secreting McAbs reactive to these key carlavirus epitopes were also developed and initially assayed; 10 cell lines were selected for further study. Potexvirus-specific PcAbs were also evaluated for broad-spectrum reactivity to peptides, virions and cloned CPs from about 20 diverse potexviruses and were shown to react with most of these potexviruses, as well as many carlaviruses. High throughput sequencing of an isolate of Tulip virus X (potexvirus) from lemon balm showing mosaic symptoms has been validated by amplification, cloning and conventional sequencing of RT-PCR products together representing most of the viral genome, including the 5⿿ and 3⿿ terminal regions. The genome sequence obtained differs from characterized isolates from tulip (Japan) or lily (Korea), likely reflecting adaptation to lemon balm, which is a dicotyledonous host in contrast to the prior monocotyledonous hosts. The genome of Helenium virus S (carlavirus) from Veronica has been partially validated by sequencing of RT-PCR products made with primers designed from the genome sequence from high throughput sequencing. In collaboration with scientists of the Central Science Laboratory, Food and Environment Research Agency, U.K. plant materials infected with Plantain virus X (PlVX) were identified, and the first ever sequence of this virus was obtained, along with a full genome sequence of two isolates by high throughput sequencing, and partial sequences of nine other isolates from the UK. A scientist from the Netherlands Food and Consumer Product Safety Authority/National Plant Protection Organization collaborated to identify and obtain partial sequences of two Dutch PlVX isolates from Plantago lanceolata. PlVX was most closely related to Actinidia virus X, reported in 2011 from Actinidia chinensis (Kiwifruit) imported to New Zealand from China. Sequence relatedness (89-100%) of the respective replicase and coat protein genes indicates that Actinidia virus X and Plantain virus X are synonymous, with the name Plantain virus X having precedence. Whereas PlVX was previously only reported from the U. K., it is now known to be present in the Netherlands in P. lanceolata, in New Zealand (and presumably China) in A. chinensis, and (as Actinidia virus X) from Canada in Ribes nigrum (Black currant). The experimental host range of PlVX includes species in at least five plant families in addition to the reported natural hosts, suggesting possible future host expansion. In partnership with Korean collaborators, infectious clones of two isolates of Tomato mosaic virus (ToMV) were produced and shown to induce symptoms of differing severity in Nicotiana benthamiana. Only two amino acid residues differentiate the proteins of each isolate, with one difference in each of the non-conserved and helicase domains of the replicase gene. Single amino acid substitutions between mild and severe symptom variants showed that the symptom differences were dependent on the residue change in the helicase domain. In partnership with Korean collaborators, infectious clones of five Turnip mosaic virus (a potyvirus) isolates from Jeju Island were produced. The viral determinants of stunting in Nicotiana benthamiana were examined by sequence comparisons and gene exchange to identify the genomic regions associated with symptom types. Four of five isolates caused mild symptoms; the fifth induced a hypersensitive response but differed by only five amino acid residues from one mild isolate. Exchange of genome fragments between these two isolates identified an amino acid residue in the HC-Pro gene that was required, but not sufficient, for induction of severe symptoms; this residue must also interact with a differential residue in the NIb gene and possibly another in the P3 gene. Variation in the P3 gene is known to influence symptoms in other potyviruses. In partnership with Korean collaborators, sequence differences between isolates of Tomato yellow leaf curl virus (TYLCV) collected in different regions of Korea in 2017 and 2018 were most closely related to previously characterized isolates belonging to either of two out of three phylogenetic clades of TYLCV, the ⿿Japan⿿ cluster or the ⿿China⿿ cluster, while some earlier-reported Korean isolates belong to a ⿿Japan+China⿿ cluster. This suggests at least three separate introductions of TYLCV into Korea, where TYLCV was first detected in 2008, and that isolates from the ⿿Japan⿿ cluster are becoming more established; only isolates of this group were detected in 2018. In partnership with Korean collaborators, interactions between the P3 and P3N-PIPO frameshift proteins of Turnip mosaic virus were examined by confocal laser scanning microscopy. Prior studies identified the P3 gene region as containing determinants of pathogenicity, but there is another essential function expressed as a frameshift protein, P3N-PIPO; differential amino acids occur in both the P3 and P3N-PIPO gene products. Fluorescently-labeled fusion proteins were expressed in Nicotiana benthamiana by agroinfiltration, and differences in subcellular localization observed between P3 and P3N-PIPO. The genomes of tree strains of Xylella fastidiosa were purified and characterized. The unique open reading frames (ORFs) of the tree, pauca and Italian olive strains were analyzed and used to develop specific qPCR tools for the detection and differentiation of pauca and Italian olive strains of X. fastidiosa (in collaboration with USDA-APHIS scientists). We isolated the first R. solanacearum-infecting phage from soil in the United States. Biological and molecular characterization of the phage were also determined. We also sequenced and characterized the first R. solanacearum-infecting phage isolated from Indonesia. We mutated five regions identified previously that contain Ralstonia solanacearum race 3 biovar 2 (r3b2)-unique DNA regions. One of the mutated regions was found to be associated with the cool virulence of r3b2, and this region was used for specific detection of r3b2 strains of R. solanacearum. Accomplishments 01 Detection of select agent strains of Ralstonia solanacearum through a DNA region associated with cool virulence. Ralstonia solanacearum r3b2 strains cause a devastating brown rot disease of potato. These bacteria are regulated select agent pathogens in the U.S. because they are capable of surviving and infecting potatoes under cool temperatures (⿿cool virulence⿿) in areas that produce seed potatoes, thereby potentially threatening U.S. agriculture. The DNA regions responsible for the cool virulence trait of R. solanacearum r3b2, however, are largely unknown and need to be identified for the accurate definition and specific detection of these select agents. ARS scientists in Beltsville, Maryland, found that when one specific DNA region of the r3b2 genome was mutated, the mutant bacterium⿿s ability to cause disease under cool, but not warm, temperature conditions was significantly reduced - suggesting the association of this region with the cool virulence of R. solanacearum r3b2. Using this information, we developed the first DNA-based detection method to target the DNA region associated with cool virulence of r3b2 for specific detection and eventual control of this important group of R. solanacearum pathogens. 02 Sequence variations affect symptoms and pathogenicity of Turnip mosaic virus. Turnip mosaic virus (TuMV) affects many food and ornamental crops; plant resistance to this virus is useful to control disease in some crops, but new isolates capable of breaking resistance often emerge. An ARS scientist in Beltsville, Maryland, collaborated with Korean scientists to generate infectious cDNA clones from 17 new radish isolates of TuMV, and differential pathogenicity and infectivity were demonstrated in radish, Chinese cabbage, and the experimental host Nicotiana benthamiana. Three classes of isolates producing only mild symptoms in N. benthamiana were identified, whereas all remaining isolates induced systemic necrosis; differential susceptibility or resistances were identified in two cultivars each of radish and Chinese cabbage, with one Chinese cabbage cultivar not infected by any isolate. Gene exchanges between a mild and a severe isolate identified a novel pathogenicity determinant allowing infection of the Chinese cabbage cultivar resistant to all of the original isolates. These results may allow breeders to identify potential new resistance genes in germplasm and their incorporation into new crop varieties. New resistance genes may be combined with other types of resistance to yield broader, longer- lasting resistance to multiple viral pathotypes. 03 Two determinants of Turnip mosaic virus are required to induce stem necrosis in Nicotiana benthamiana. Introduction of novel viral pathotypes into a new area may cause breakdown of crop resistance. An ARS scientist in Beltsville, Maryland, collaborated with Korean scientists to determine whether sequence differences between four radish isolates of Turnip mosaic virus from China were associated with the degree of symptom severity. Only two isolates induced obvious symptoms in the natural host (radish), whereas symptoms in an experimental host (Nicotiana benthamiana) varied from mild to severe mosaic with associated stem necrosis. A domain containing three of four differential amino acid residues distinguishing two isolates conferred most of the more severe symptom characteristics, but not stem necrosis, in Nicotiana benthamiana. An interaction between a particular differential residue in this domain and the fourth differential residue in a different domain is most likely required to induce stem necrosis, as neither alone was sufficient. These isolates induced differential symptom severity in two radish cultivars, indicating that the radish genotype itself affects symptom severity. Similarities between the Chinese isolates and previously characterized Korean isolates suggests genetic interchange between Chinese and Korean isolates through trade, or as a result of long-distance transmission by aphid vectors carried by prevailing winds. 04 Molecular and biological characterization of the first Ralstonia phage, RsoM1USA, isolated from soil in the United States. Ralstonia bacteriophages are viruses that specifically infect the bacterium Ralstonia solanacearum (Rs), a causal agent of a destructive bacterial wilt disease in tropical, subtropical and warm temperate regions of the world. They have recently been isolated from soils in Japan, Thailand, Korea and Egypt. In an effort to better understand their relationships with Rs and to determine their potential as biocontrol agents for Rs, ARS scientists in Beltsville, Maryland, purified and characterized RsoM1USA, the first Ralstonia-infecting phage isolated from soil in the United States. Infection of Ralstonia by phage RsoM1USA resulted in significantly reduced in vitro growth of the infected bacterium, but not disease symptoms caused by the bacterium in tomato plants. Our goal is to determine if RsoM1USA offers a competitive advantage to the infected bacterial strain for persistence of the bacterium in the environment. 05 Detection and first report of beet ringspot virus in ornamental Oxalis in the United States. Ornamental Oxalis triangularis, commonly known as False Shamrock because of its triangular leaves, is grown as a potted plant in the United States, especially for marketing in the spring around St. Patrick⿿s Day. ARS scientists in Beltsville, Maryland, discovered a previously unreported virus in Oxalis plants from Wisconsin that were showing chlorotic ringspot symptoms. Full genome sequence analysis revealed that this virus was a strain of beet ringspot virus (BRSV), a soil-borne virus originally thought to be synonymous with the quarantine-regulated nepovirus tobacco blackring virus. BRSV infects a wide range of plant species including potato, sugar beet, strawberry, turnip, wheat, oat, peach, begonia and Euonymus. This is the first report of BRSV in ornamental Oxalis. The information and detection tools developed in this work will be useful to ornamental nurseries, public and private plant disease diagnostic clinics, and other scientists who study ornamental viruses, as well as to state and federal regulatory officials to help them make timely and appropriate recommendations in safeguarding the movement of horticultural products into and throughout the U.S.
Impacts
(N/A)
Publications
- Addy, H.S., Farid, M.M., Ebrahim, A., Huang, Q. 2018. Host range and molecular characterization of a lytic Pradovirus-like Ralstonia phage RsPod1IDN isolated from Indonesia. Archives of Virology. 163:3409-3414.
- Addy, H.S., Ebrahim, A., Huang, Q. 2019. Molecular and biological characterization of Ralstonia phage RsoM1USA, a new species of P2Virus, isolated in the USA. Frontiers in Microbiology. 10:267.
- Dey, K.K., Leite, M., Hu, J.S., Jordan, R.L., Melzer, M.J. 2018. Detection of Jasmine virus H and characterization of a second pelarspovirus infecting star jasmine (Jasminum multiflorum) and angelwing jasmine (J. nitidum) plants displaying virus-like symptoms. Archives of Virology. 163:3051-3058.
- Gong, J., Ju, H., Kim, I., Seo, E., Cho, I., Han, J., Kim, J., Lim, Y., Hammond, J., Lim, H. 2019. Sequence variations among 17 new radish isolates of turnip mosaic virus showing differential pathogenicity and infectivity in Nicotiana benthamiana, Brassica rapa, and Raphanus sativus. Phytopathology. 109:904-912.
- Byrne, D.H., Klein, P., Hall, C., Windham, M., Ochoa-Corona, F., Olson, J., Paret, M., Babu, M., Knox, G., Jordan, R.L., Hammond, J., Ong, K., Ochoa, R., Bauchan, G.R., Evans, T., Windham, A., Hale, F., Palma, M.A., Ribera, L., Pemberton, H.B. 2019. Combating rose rosette disease US national project. Acta Horticulturae. 1232:203-212.
- Cho, I., Chung, B.N., Hammond, J., Moon, J., Lim, H. 2018. First report of Grapevine rupestris vein feathering virus infecting grapevines in Korea. Plant Disease. 102:1471.
- Otero-Colina, G., Ochoa, R., Amrine, J., Hammond, J., Jordan, R.L., Bauchan, G.R. 2019. Eriophyoid mites found on roses in the United States. Journal of Environmental Horticulture. 36(4):146-153.
- Bauchan, G.R., Otero-Colina, G., Hammond, J., Jordan, R.L., Ochoa, R. 2019. Rose rosette disease: It all started with a small mite. Acta Horticulturae. 1232(33):227-232.
- Ju, H., Kim, I., Hu, W., Kim, B., Choi, G., Kim, J., Lim, Y., Domier, L.L., Hammond, J., Lim, H. 2019. A single nucleotide change in the overlapping MP and CP reading frames results in differences in symptoms caused by two isolates of Youcai mosaic virus. Archives of Virology. 164(6):1553-1565.
- Kim, B., Cho, I., Kim, I., Choi, G., Ju, H., Hu, W., Oh, J., Kim, J., Seo, E., Domier, L.L., Hammond, J., Lim, H. 2019. Length of poly(A) tail affects transcript infectivity of three ZYMV symptom variants differing at only five animo acid positions. Journal of Plant Pathology.
- Kim, I., Ju, H., Gong, J., Han, J., Seo, E., Cho, S., Hu, W., Choi, S., Lim, Y., Domier, L.L., Hammond, J., Lim, H. 2019. A Turnip mosaic virus determinant of systemic necrosis in Nicotiana benthamiana, and a novel resistance-breaking determinant in Chinese cabbage identified from chimeric infectious clones. Phytopathology. 109:1638-1647.
- Hu, W., Seo, E., Cho, I., Kim, J., Ju, H., Kim, I., Choi, G., Kim, B., Ahn, C., Domier, L.L., Hammond, J., Lim, H. 2019. Amino acid differences in the N-terminal half of the polyprotein of Chinese turnip mosaic virus isolates affect symptom expression in Nicotiana benthamiana and radish. Archives of Virology. 164(6):1683-1689.
- Jordan, R.L., Mollov, D.S., Guaragna, M., Lockhart, B. 2019. Detection and first report of Beet ringspot virus in ornamental Oxalis in the United States. Plant Disease. 103:1800.
- Stulberg, M.J., Cai, X., Ebrahim, A., Huang, Q. 2018. Identification of a DNA region associated with the cool virulence of Ralstonia solancearum strain UW551 and its utilization for specific detection of the bacterium⿿s race 3 biovar 2 strains. PLoS One. 13(11).
- Cho, I., Chung, B., Kwon, S., Yoon, J., Choi, G., Kim, B., Kwak, Y., Hammond, J., Lim, H. 2019. First report of Zucchini yellow mosaic virus in muskmelon (Cucumis melo) in Korea. Journal of Plant Pathology. 101(3):771.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): The three objectives of this project are: (1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1]; (2) Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. [NP303, C2, PS2A]; and, (3) Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. Identify and characterize genes and/or phages affecting virulence and competitiveness of those bacteria to develop effective control methods. [NP303, C1, PS1]. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants (tested for absence of specific pathogens) for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Approach (from AD-416): The overall approach is to develop knowledge, tools, and reagents to aid U.S. floricultural producers and diagnosticians to establish and apply effective virus testing protocols to improve clean stock production for vegetatively-propagated annuals and perennials. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Based on the knowledge and tools developed while identifying and characterizing new viruses and comparisons to previously-characterized viruses, new virus-specific and broad spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, nucleic acid hybridization probes, PCR primers, isothermal amplification methods, and improved associated protocols will be developed. Validation of the recently devloped Universal Plant Virus Microarray (UPVM) will continue in order to transfer the UPVM technology to potential users. Next generation sequencing (NGS) of nucleic acid extracts from plants infected with unknown viruses is expected to yield information about the genomes of previously uncharacterized viruses without any background information on what viruses might be infecting the plant. Both NGS and UPVM have the potential to identify any virus present and identify all components of mixed infections, and is suited to application in situations where rapid results are important (in Quarantine operations and germplasm introduction). Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. We will make modifications to infectious clones of selected viruses by gene exchange and site-directed mutagenesis. We will examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST- pulldown assays. VIGS and/or protein over-expression will also be utilized. Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. The genomic DNA sequences of ornamental strains of Xylella fastidiosa (Xf) will be determined. The genetic diversity and phylogenetic relatedness among woody ornamental and non-ornamental strains will be evaluated. This sequence information will be used to develop specific PCR detection tools for woody ornamental strains of Xf. The identification and characterization of genes and regulatory elements, including phages, affecting virulence and/or competitiveness of Ralstonia solanacearum (including Race 3 Biovar 2) will be studied. This information will be used to further develop accurate detection tools and effective control methods. Objective 1a: We have continued to examine samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples included a plant of Zizia aurea received from the Missouri Department of Agriculture, showing fine chlorotic spotting on the foliage; electron microscopy of embedded leaf material revealed the presence of paracrystalline arrays of small isometric virus particles, but not yet further identified; and, a nucleic acid extract of symptomatic leaf tissue has been processed for high throughput sequencing (HTS). Total RNAs from several gardenia plants from Deleware and Mar exhibiting chlorotic ringspot and line pattern symptoms were prepared for HTS. Onion yellow dwarf virus was detected in an ornamental Allium plant for the first time. No virus was detected in samples of beech trees from Ohio affected by decline, by either electron microscopy of leaf extracts or embedded tissue, or by HTS. In collaboration with scientists from the Systematic Entomology Laboratory and the Electron and Confocal Microscopy Unit at Beltsville, Maryland, we continue to examine the interactions between eriophyid mites and different rose species and genotypes. Material from additional rose varieties has been received from collaborators at several universities as part of a USDA-NIFA-SCRI project to examine the viral, vector, and host factors influencing the spread of Rose rosette virus and its ability to cause disease in roses. In collaboration with a visiting scientist from India, we initiated development of both serological and nucleic acid-based (RT-RPA) lateral flow assays for detection of Plantago asiatica mosaic virus (PlAMV). Specific detection of PlAMV was achieved by both methods, but further optimization of materials and extraction conditions is required to increase the sensitivity of the serological lateral flow assay. Improved rapid extraction methods are necessary for effective utilization of the RT-RPA lateral flow assay. In partnership with collaborators in Costa Rica, Plantago asiatica mosaic virus was detected in imported lilies grown in Costa Rica, the first time the presence of PlAMV was confirmed in that country. Objective 1b: We have continued to utilize existing broad-spectrum PCR primers for analysis of a wide range of suspected virus-infected plants, allowing the detection of previously reported viruses in new hosts, or obtaining products for sequence analysis of newly-discovered viruses allowing their comparison to previously characterized viruses. Using these generic primers, amplified products were obtained and sequenced from a variety of ornamental plant species, including narcissus (a potyvirus, a potexvirus, and a carlavirus from a single plant), veronica (the carlavirus HelVS), and lemon balm (the potexvirus TVX). In our ongoing research on the development of broad-spectrum reacting antibodies to important plant virus species, rabbit polyclonal antibodies (PcAbs) have been generated to synthetic peptide immunogens representing highly conserved carlavirus antigenic sites on viral coat proteins. Hybridoma cell lines secreting mouse monoclonal antibodies (McAbs) reactive to key epitopes are being developed. We will screen the PcAb and McAbs against the immunogen peptides, plus virions and bacterially- expressed cloned CPs from our current collection of more than 20 diverse carlaviruses. Synthetic peptides representing highly conserved potexvirus antigenic coat protein sites have also been used as immunogen for rabbit PcAb production. These will be evaluated for their broad-spectrum reactivity to purified virions and expressed cloned CPs from our current collection of about 20 diverse potexviruses. In ongoing collaborative research (a USDA-NIFA-SCRI Project) to develop efficient serological diagnostic tools to enable the rapid, user-friendly and accurate detection of Rose rosette virus (RRV), our previously developed RRV NP-specific rabbit PcAbs and ten selected McAbs were shown to detect RRV antigen in Western-blots and several ELISA formats. Twelve synthetic peptides representing predicted antigenic sites on the NP were used to define the epitope-specificity of the PcAb and McAbs. These tests showed that using the PcAb as an ELISA-trapping antibody and an admixture of two McAbs as the detecting antibodies would predictably be the best format for the detection of RRV in plants. This is currently being tested with healthy and RRV-infected rose samples from across the US. Objective 1d: The full genome sequence of a HelVS isolate from a cultivar of Veronica was determined by Illumina MiSeq high throughput sequencing (HTS); this represents the first full genome sequence of this carlavirus, which is known to infect several species of ornamentals, including Helenium and Impatiens, as well as Veronica. The full genome sequence of an isolate of Tulip virus X was also obtained by HTS from a lemon balm plant showing mosaic symptoms. The partial genome sequence of an isolate of Apple mosaic virus was determined from a highly symptomatic sample of Magnolia tripetala. Total RNA from a Callicarpa americana beautyberry plant growing in Alabama and exhibiting mosaic symptoms was analyzed by HTS. Viral sequences were identified that had significant putative protein homologies with members of the genus Emaravirus. Complete or near-complete sequences were obtained for genomic RNAs 1-4. HTS and RT-PCR analysis of other Callicarpa samples is ongoing. To our knowledge, this is the first report of an emaravirus infecting Callicarpa. Objective 2a: In partnership with Korean collaborators, full length infectious clones of two isolates of the tobamovirus Youcai mosaic virus (YoMV) were produced and shown to induce symptoms of differing severity in Nicotiana benthamiana. Sequence comparisons and subsequent gene exchange between the mild and severe symptom variants demonstrated that the symptom differences were dependent on a single residue change in the coat protein gene. Full length infectious clones of three isolates of Zucchini yellow mosaic virus differing in symptom severity were also produced in partnership with Korean collaborators, with efficiency of infectivity, but not the symptom differences, found to depend on the length of the 3'- terminal poly(A) tail, with much greater infectivity when the length was increased from about 30 to 60 residues. Objective 2b: We demonstrated that specific sequences of the Lolium latent virus coat proteins, which are unique to this type of virus, target specific sequences of proteins associated with chloroplasts of the plant cell. It is this interaction that ultimately facilitates the transfer and spread of the virus. This work identifies a method by which the virus spreads through the host plant which virologists, pathologists and disease managers can target as the first step in developing countermeasures for mitigating the disease and thus reducing disease losses in agriculturally important plants. In partnership with Korean collaborators, interactions between the two coat proteins and the movement protein of Radish mosaic virus were examined, together with their subcellular localization. When expressed individually as fluorescent fusion proteins, and examined by confocal microscopy, the movement protein (MP) was localized at the cell periphery, whereas the large coat protein (LCP) subunit accumulated as punctate aggregates in the cytoplasm, and the small coat protein (SCP) subunit was distributed throughout the cytoplasm and at the cell periphery. Results from co-expression of free and fluorescently-labeled proteins showed that SCP can interact separately with both LCP and MP, and that MP determines the localization of combinations. In collaboration with a Japanese scientist, the localization patterns and RNA silencing suppression efficiency of variants of the TGB1 protein of a lily isolate of Plantago asiatica mosaic virus (PlAMV) were examined. As we have previously demonstrated with other potexvirus TGB1 proteins, certain amino acid substitutions affect the ability of the TGB1 protein to localize to the nucleus and nucleolus, and these variants also differ in their efficacy of RNA silencing suppression. Objective 3.1: We propagated and verified a tree strain of X. fastidiosa isolated from Virginia, and prepared genomic DNAs and sent the DNAs for sequencing. We compared these genomic sequences to other landscape tree strains of X. fastidiosa that we had sequenced before or identified in GenBank for conservation of unique genes and phylogenetic relationships. We also searched GenBank for potential functions of the 150 different R. solanacearum race 3 biovar 2-unique DNA regions identified previously and were able to cluster these into 32 regions in the genome of R. solanacearum as potential targets for mutagenesis. Objective 3.2: We isolated a novel Ralstonia phage from soil in Egypt and have designated it Ralstonia phage RsoP1EGY. This phage specifically infects only r3b2 phylotype IIB sequevar 1, and not non-r3b2 strains of R. solanacearum and has properties similar to other podovirus phages. The 41 kbp genome shares no significant sequence identity to any other reported R. solanacearum or non-Ralstonia phages and is the first sequenced and characterized R. solanacearum phage isolated in Egypt. It has potential as a biocontrol agent for r3b2 strains of R. solanacearum in all countries where potato brown rot is a problem. We determined the effect of another phage, Rs551, on its carrier R. solanacearum strain UW551 by deleting 60% of the phage�s prophage region from the UW551 genome. The deletion resulted in significantly increased virulence as compared to the wild type UW551. Our results suggest that phage Rs551 may play an important ecological role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment. Accomplishments 01 A datasheet on Plantago asiatica mosaic virus added to Invasive Species Compendium. Plantago asiatica mosaic virus is a rapidly emerging pathogen first discovered in the mid 1970s in the Russian Far East that is affecting multiple crops throughout the world, including wild- growing and cultivated lilies. In lilies, the virus is thought to be spread through the international bulb trade, although the original source of infection is not known. An ARS scientist in Beltsville, Maryland, published a datasheet that presents a summary of what is known of the basic biology of the virus, including both natural and experimental host range, transmission in the absence of any known biotic vector, and methods which may be useful to control infection. This information will be useful to quarantine officials, producers, and consumers to prevent the spread to new crops and countries where the virus is not yet established, and to reduce disease losses in agriculturally and horticulturally important crops. 02 Discovery of novel pelarspoviruses infecting jasmine from Hawaii, DC, Maryland, and California. A myriad of foliar symptoms, including ringspots, line patterns, mosaic, mottling, and leaf deformation were observed on species of jasmine plants growing in Hawaii and at the U.S. National Arboretum in Washington, DC. ARS scientists in Beltsville, Maryland, discovered two distinct pelarspovirus species (called JaVH and JMaV) infecting these plants, and developed an assay to detect and differentiate these virus species in jasmine plants from other locations. These assays revealed that both virus species were found in samples tested from Hawaii, DC, and Maryland, but that only one species (JMaV) was detected in a symptomatic sample from California. This information will increase our understanding of the genetic diversity of these two new viruses and will be useful to ornamental nurseries, public and private plant disease diagnostic clinics, and other scientists who study ornamental viruses. 03 Prophage from select agent Ralstonia solanacearum affects virulence of this bacteria. R. solanacearum is a bacterial species that causes millions of dollars of crop losses in a wide range of plant species worldwide. One strain in particular, the r3b2 subgroup, is such a threat to U.S. agriculture that it has been designated a select agent and is subject to strict quarantine regulations. ARS scientists in Beltsville, Maryland, characterized a filamentous lysogenic bacteriophage (named Rs551) that is stably maintained as a prophage in the genome of a r3b2 strain. Infection of the Rs551 phage into a phage- free strain of R. solanacearum significantly reduced the pathogen�s virulence, while deletion of the phage genome from a naturally-infected r3b2 strain resulted in significantly increased virulence compared to the wild type strain. The fact that this phage genome was also present in the genomes of 11 other r3b2 strains suggests an evolutionary and biological role as well as a possible target to manage and control this important pathogen.
Impacts
(N/A)
Publications
- Vaira, A., Lim, H., Bauchan, G.R., Miozzi, L., Vinals, N., Natilla, A., Owens, R.A., Hammond, J. 2018. The interaction of Lolium latent virus major coat protein with ankyrin repeat protein NbANKr redirects it to chloroplasts and modulates virus infection. Plant Journal. 99(5):730-742.
- Garcia, M., Dal Bo, E., Da Graca, J., Gago-Zachert, S., Hammond, J., Moreno, P., Natsuaki, T., Pallas, V., Navarro, J., Reyes, C., Robles Luna, G., Sasaya, T., Tzanetakis, I., Vaira, A., Verbeek, M. 2017. Family Ophioviridae: classification and features. Journal of General Virology. 98(6):1161-1162.
- Kim, N., Seo, E., Han, S., Gong, J., Domier, L.L., Hammond, J., Jang, C., Lim, H. 2017. Pseudomonas oleovorans strain KBPF-004 culture supernatants reduced seed transmission of Cucumber green mottle mosaic virus and Pepper mild mottle virus, and remodeled aggregation of 126 kDa and subcellular localization of movement protein of Pepper mild mottle virus. Plant Pathology Journal. 33(4):393-401.
- Kim, I., Han, J., Cho, I., Moon, J., Seo, E., Kim, H., Hammond, J., Lim, H. 2017. Generation of an infectious clone of a new Korean isolate of apple chlorotic leaf spot virus (ACLSV) driven by dual 35S and T7 promoters in a versatile binary vector. Virus Genes. 33(6):608-613.
- Ebrahim, A., Stulberg, M.J., Mershon, J.P., Mollov, D.S., Huang, Q. 2017. Molecular and biological characterization of 'Rs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum. PLoS One.
- Cho, I., Kim, S., Kwon, S., Chung, B., Hammond, J., Lim, H. 2018. First report of a typical calico-associated isolate Peach latent mosaic viroid from calico disease-affected peach trees in Korea. Plant Disease.
- Cho, I., Kwon, S., Yoon, J., Chung, B., Hammond, J., Lim, H. 2017. First report of Apple necrotic mosaic virus infecting apple trees in Korea. Journal of Plant Pathology. 99(3):815. Available:
- Jordan, R.L., Wingert, M., Louden, C., Guaragna, M. 2018. First report of nerine latent virus in ornamental crinum in the United States. Plant Disease. 102:1469. doi.org/10.1094/PDIS-09-17-1512-PDN.
- Ebrahim, A., Stulberg, M.J., Huang, Q. 2017. Prophage Rs551 and its repressor gene orf14 reduce virulence and increase competitive fitness of its Ralstonia solanacearum carrier strain UW551. Frontiers in Microbiology. 8:2480.
- Ebrahim, A., Elhalag, K.M., Addy, H., Hussien, A.S., Nasr-Eldin, M.A., Huang, Q. 2018. Sequencing, genome analysis and host range of a novel Ralstonia phage RsoP1EGY isolated from Egypt. Archives of Virology.
- Hammond, J. 2018. Plantago asiatica mosaic virus � data sheet. Center for Agriculture and Biosciences International (CABI) Invasive Species Compendium. Available:
- Choi, G., Kim, B., Ju, H., Seo, E., Kim, J., Park, J., Hammond, J., Lim, H. 2018. Dual infections of ToMV and TYLCV, or ToMV and ToCV, detected in tomato fields located in Chungchungnam-Do in 2017. Korean Journal of Agricultural Science. 45(1):38-42.
- Montero-Astua, M., Garita, L., Vasquez, E., Hammond, J., Moreiera, L. 2017. Detection of Plantago asiatica mosaic virus in lily hybrid plants (Lilium spp.) in Costa Rica grown from imported bulbs. Australasian Plant Disease Notes. 12:47.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): The three objectives of this project are: (1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1]; (2) Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. [NP303, C2, PS2A]; and, (3) Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. Identify and characterize genes and/or phages affecting virulence and competitiveness of those bacteria to develop effective control methods. [NP303, C1, PS1]. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants (tested for absence of specific pathogens) for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Approach (from AD-416): The overall approach is to develop knowledge, tools, and reagents to aid U.S. floricultural producers and diagnosticians to establish and apply effective virus testing protocols to improve clean stock production for vegetatively-propagated annuals and perennials. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Based on the knowledge and tools developed while identifying and characterizing new viruses and comparisons to previously-characterized viruses, new virus-specific and broad spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, nucleic acid hybridization probes, PCR primers, isothermal amplification methods, and improved associated protocols will be developed. Validation of the recently devloped Universal Plant Virus Microarray (UPVM) will continue in order to transfer the UPVM technology to potential users. Next generation sequencing (NGS) of nucleic acid extracts from plants infected with unknown viruses is expected to yield information about the genomes of previously uncharacterized viruses without any background information on what viruses might be infecting the plant. Both NGS and UPVM have the potential to identify any virus present and identify all components of mixed infections, and is suited to application in situations where rapid results are important (in Quarantine operations and germplasm introduction). Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. We will make modifications to infectious clones of selected viruses by gene exchange and site-directed mutagenesis. We will examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST- pulldown assays. VIGS and/or protein over-expression will also be utilized. Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. The genomic DNA sequences of ornamental strains of Xylella fastidiosa (Xf) will be determined. The genetic diversity and phylogenetic relatedness among woody ornamental and non-ornamental strains will be evaluated. This sequence information will be used to develop specific PCR detection tools for woody ornamental strains of Xf. The identification and characterization of genes and regulatory elements, including phages, affecting virulence and/or competitiveness of Ralstonia solanacearum (including Race 3 Biovar 2) will be studied. This information will be used to further develop accurate detection tools and effective control methods. This new project began on 04/09/2017 and represents a continuation of Project 8020-222000-032-00D. Progress towards milestones and objectives for FY17 is reported in the report for the terminating project. Please see the report for 8020-22000-032-00D for detailed information.
Impacts
(N/A)
Publications
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